Antileishmanial dinitroaniline sulfanomides with activity against parasite tubulin

ABSTRACT

Dinitroaniline compounds useful for the treatment of diseases caused by parasitic protozoa in subjects in need of such treatment. The compounds are particularly useful in the treatment of leishmaniasis. The compounds are preferably less cytotoxic to normal cells than oryzalin. Also provided are methods of treating subjects having diseases caused by parasitic protozoa, preferably humans. The method comprising administering a therapeutically effective amount of a dinitroaniline compound of the present invention to a subject in need of such treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationsNos. 60/374,727 filed Apr. 23, 2002, and 60/382,965 filed May 24, 2002,the entirety of each are incorporated herein by reference.

BACKGROUND OF THE INVENTION

There is a general lack of effective, inexpensive chemotherapeuticagents for the treatment of parasitic protozoal diseases that occur inthe developing world. These parasitic protozoal diseases includeleishmaniasis, including visceral leishmaniasis, mucocutaneousleishmaniasis, and cutaneous leishmaniasis; Chagas disease; humanAfrican trypanosomiasis, also known as African sleeping sickness; animaltrypanosomiasis; and malaria.

Leishmaniasis currently threatens 350 million men, women and children in88 countries around the world. The leishmaniases are parasitic diseaseswith a wide range of clinical symptoms: cutaneous, mucocutaneous andvisceral. Visceral leishmaniasis—also known as kala azar ischaracterized by irregular bouts of fever, substantial weight loss,swelling of the spleen and liver, and anemia (occasionally serious). Ifleft untreated, the fatality rate can be as high as 100%. Inmucocutaneous forms of leishmaniasis, lesions can lead to partial ortotal destruction of the mucose membranes of the nose, mouth and throatcavities and surrounding tissues. These disabling and degrading forms ofleishmaniasis can result in victims being humiliated and cast out fromsociety. Cutaneous forms of the disease normally produce skin ulcers onthe exposed parts of the body such as the face, arms and legs. Thedisease can produce a large number of lesions-sometimes up to200-causing serious disability and invariably leaving the patientpermanently scarred, a stigma which can cause serious social prejudice.

The leishmaniases are caused by different species belonging to the genusLeishmania a protozoa transmitted by the bite of a tiny 2 to 3millimeter-long insect vector, the phlebotomine sandfly. Of 500 knownphlebotomine species, only some 30 of them have been positivelyidentified as vectors of the disease. Only the female sandfly transmitsthe protozoan, infecting itself with the Leishmania parasites containedin the blood it sucks from its human or mammalian host in order toobtain the protein necessary to develop its eggs. During a period of 4to 25 days, the parasite continues its development inside the sandflywhere it undergoes major transformation. When the now infectious femalesandfly feeds on a fresh source of blood, its painful sting inoculatesits new victim with the parasite, and the transmission cycle iscompleted.

The insect vector of leishmaniasis, the phlebotomine sandfly, is foundthroughout the world's inter-tropical and temperate regions. The femalesandfly lays its eggs in the burrows of certain rodents, in the bark ofold trees, in ruined buildings, in cracks in house walls, and inhousehold rubbish, as it is in such environments that the larvae willfind the organic matter, heat and humidity which are necessary to theirdevelopment. In its search for blood (usually in the evening and atnight), the female sandfly covers a radius of a few meters to severalhundreds around its habitat. For a long time, little was known about thetransmission cycles of the disease, but over the last few years, fieldresearch and the application of molecular biology have enabledsubstantial progress to be made in understanding the different links inthe transmission chain. Moreover, simple new diagnostic techniques haverecently been developed which are practical, reliable and inexpensive.These techniques are available to concerned countries for the earlydetection and rapid treatment of the disease.

The Leishmaniases are related to environmental changes such asdeforestation, building of dams, new irrigation schemes, urbanizationand migration of non-immune people to endemic areas. It seriouslyhampers productivity and vitally needed socioeconomic progress andepidemics have significantly delayed the implementation of numerousdevelopment programs. This is particularly true in Saudi Arabia,Morocco, the Amazon basin and the tropical regions of the Andeancountries.

For many years, the public health impact of the leishmaniases has beengrossly underestimated, mainly due to lack of awareness of its seriousimpact on health. Over the last 10 years endemic regions have beenspreading further afield and there has been a sharp increase in thenumber of recorded cases of the disease. As declaration is obligatory inonly 32 of the 88 countries affected by leishmaniasis, a substantialnumber of cases are never recorder. In fact, of the 1.5-2 million newcases estimated to occur annually, only 600,000 are officially declared

In addition, deadly epidemics of visceral leishmaniasis periodicallyflare up. For example, in the 1990s Sudan suffered a crisis with anexcess mortality of 100,000 deaths among people at risk. An epidemic ofcutaneous leishmaniasis is ongoing in Kabul, Afghanistan with anestimated 200,000 cases.

Chagas disease has a wide distribution in Central and South America,being found only in the American Hemisphere. It is endemic in 21countries, with 16-18 million persons infected and 100 million people atrisk. The disease is caused by Trypanosoma cruzi, a flagellatedprotozoan parasite which is transmitted to humans in two ways, either bya blood-sucking reduviid bug which deposits its infective feces on theskin at the time of biting, or directly by transfusion of infectedblood. Humans and a large number of species of domestic and wild animalsconstitute the reservoir, and the vector bugs infest poor housing andthatched roofs.

The acute stage of the disease is generally seen in children, and ischaracterized by fever, swelling of lymph glands, enlargement of theliver and spleen, or local inflammation at the site of infection. But,commonly, there are no acute clinical manifestations, and those infectedmay remain without symptoms. In about one-third of acute cases, achronic form develops some 10-20 years later, causing irreversibledamage to the heart, esophagus and colon, with dilatation and disordersof nerve conduction of these organs. Patients with severe chronicdisease become progressively more ill and ultimately die, usually fromheart failure. There is, at present, no effective treatment for suchcases.

Rural migrations to urban areas during the 1970s and 1980s changed thetraditional epidemiological pattern of Chagas disease: it became anurban disease, as unscreened blood transfusion created a second way oftransmission. Between 1960 and 1989, the prevalence of infected blood inblood banks in selected cities of South America ranged from 1.7% in SaoPaulo, Brazil to 53.0% in Santa Cruz, Bolivia, a percentage far higherthan that of hepatitis or HIV infection.

Human African trypanosomiasis, known as sleeping sickness, is avector-borne parasitic disease. Trypanosoma, the parasites concerned,are protozoa transmitted to humans by tsetse flies (glossina). Tsetseflies live in Africa, and they are found in vegetation by rivers andlakes, gallery-forests and vast stretches of wooded savannah. Sleepingsickness occurs only in sub-Saharan Africa, in regions where tsetseflies are endemic. For reasons as yet unknown, there are many regionswhere tsetse flies are found, but sleeping sickness is not. The ruralpopulations that live in such environments and depend on them foragriculture, fishing, animal husbandry or hunting are the mostexposed-along with their livestock-to the bite of the tsetse fly.

Sleeping sickness affects remote and rural areas where health systemsare least effective, or non-existent. It spreads with socio-economicproblems such as political instability, displacement of populations, warand poverty. It develops in foci whose size can range from a village toan entire region. Within a given focus, the intensity of the disease canvary considerably from one village to the next.

Human African trypanosomiasis takes two forms, depending on the parasiteinvolved: Trypanosoma brucei gambiense (T.b. gambiense) is found incentral and West Africa. It causes chronic infection, which does notmean benign. A person can be infected for months or even years withoutobvious symptoms of the disease emerging. When symptoms do emerge, thedisease is already at an advanced stage. Trypanosoma brucei rhodesiense(T.b. rhodesiense) is found in southern and east Africa. It causes acuteinfection that emerges after a few weeks. It is more virulent than theother strain and develops more rapidly, which means that it is morequickly detected clinically.

Other sub-species of the parasite cause animal trypanosomiasis, whichare pathogenic to animals and are often different from those that causethe disease in humans. Animals can carry parasites, especially T.b.rhodesiense; domestic and wild animals are a major reservoir. They canalso be infected with T. b. gambiense, though the precise role of thisreservoir is not well known. The two human and animal forms of thedisease remain a major obstacle to the development of rural regions ofsub-Saharan Africa: human loss, decimation of cattle and abandonment offertile land where the disease is rife.

There have been three severe epidemics in Africa over the last century:one between 1896 and 1906, mostly in Uganda and the Congo Basin, one in1920 in several African countries, and one that began in 1970 and isstill in progress. The 1920 epidemic was arrested due to mobile teamssystematically screening millions of people at risk. The disease hadpractically disappeared between 1960 and 1965. After that success,screening and effective surveillance were relaxed, and the disease hasreappeared in endemic form in several foci over the last thirty years.

Sleeping sickness threatens over 60 million people in 36 countries ofsub-Saharan Africa. Only 3 to 4 million people at risk are undersurveillance, with regular examination or access to a health centre thatcan provide screening. Detection of the disease calls for major humanand material resources, such as well-equipped health centers andqualified staff. Because such resources are lacking, most people withsleeping sickness die before they can ever be diagnosed. Almost 45,000cases were reported in 1999, but the World Health Organization (WHO)estimates that the number of people affected is ten times greater. The45,000 case figure shows not the true situation but rather the lack ofscreening in many foci. The real number of cases seems to be between300,000 and 500,000. Reported cases in recent years are from countrieswhere surveillance coverage is no more than 5%.

In certain villages of many provinces of Angola, the Democratic Republicof Congo and southern Sudan, the prevalence is between 20% and 50%.Sleeping sickness has become the first or second greatest cause ofmortality, ahead of HIV/AIDS, in those provinces.

Countries are placed in four categories in terms of prevalence. In eachcountry the spatial distribution of the disease is very diverse; it isfound in foci and micro-foci. Countries where there is an epidemic ofthe disease, in terms of very high cumulated prevalence and hightransmission: Angola, Democratic Republic of Congo and Sudan. Highlyendemic countries, where prevalence is moderate but increase is certain:Cameroon, Central African Republic, Chad, Congo, Côte d'Ivoire, Guinea,Mozambique, Uganda and United Republic of Tanzania. Countries where theendemic level is low: Benin, Burkina Faso, Equatorial Guinea, Gabon,Kenya, Mali, Togo and Zambia. Countries whose present status is notclear: Botswana, Burundi, Ethiopia, Liberia, Namibia, Nigeria, Rwanda,Senegal and Sierra-Leone.

The disease is transmitted with the bite of the tsetse fly. At first thetrypanosomes multiply in the blood, and that process can last for yearswith T.b. gambiense. Mother-to-child infection: the trypanosome cancross the placenta and infect the fetus, causing abortion and perinataldeath. Accidental infections can occur in laboratories, for example,through the handling of blood of an infected person, although this isfairly rare. The early phase entails bouts of fever, headaches, pains inthe joints and itching. The second, known as the neurological phase,begins when the parasite crosses the blood-brain barrier and infests thecentral nervous system. This is when the characteristic signs andsymptoms of the disease appear: confusion, sensory disturbances and poorcoordination. Disturbance of the sleep cycle, which gives the diseaseits name, is the most important feature. Without treatment, the diseaseis fatal. If the patient does not receive treatment before the onset ofthe second phase, neurological damage is irreversible even aftertreatment.

There are three stages to case management: Screening is the initialsorting of people who might be infected. This involves checking forclinical signs or the use of serological tests. Diagnosis shows whetherthe parasite is present. The only sign, one that has been known forcenturies, is swollen cervical glands. Phase diagnosis shows the stateof progression of the disease. It entails examination of cerebro-spinalfluid obtained by lumbar puncture and is used to determine the course oftreatment. The long, asymptomatic first phase of T.b. gambiense sleepingsickness is one of the factors that makes treatment difficult. Diagnosismust be made as early as possible in order to preclude the onset ofirreversible neurological disorders and prevent transmission. Casedetection is difficult and requires major human, technical and materialresources. Since the disease is rife in rural areas among poor peoplewith little access to health facilities, this problem is all the moredifficult.

If the disease is diagnosed early, the chances of cure are high, butearly diagnosis of the disease, which would guarantee low-risk treatmenton an outpatient basis, can rarely be achieved. The type of treatmentdepends on the phase of the disease: initial or neurological. Success inthe latter phase depends on having a drug that can cross the blood-brainbarrier to reach the parasite. Four drugs have been used until now.However drugs are old, difficult to administer in poor conditions and byno means always successful.

Malaria is a life-threatening parasitic disease transmitted bymosquitoes. Today approximately 40% of the world's population—mostlythose living in the world's poorest countries—is at risk of malaria. Thedisease was once more widespread but it was successfully eliminated frommany countries with temperate climates during the mid 20th century.Today malaria is found throughout the tropical and sub-tropical regionsof the world and causes more than 300 million acute illnesses and atleast one million deaths annually. Ninety per cent of deaths due tomalaria occur in Africa, south of the Sahara—mostly among youngchildren. Malaria kills an African child every 30 seconds. Many childrenwho survive an episode of severe malaria may suffer from learningimpairments or brain damage. Pregnant women and their unborn childrenare also particularly vulnerable to malaria, which is a major cause ofperinatal mortality, low birth weight and maternal anemia.

Malaria parasites have become resistant to one drug after another andmany insecticides are no longer useful against the mosquitoes whichtransmit the disease. Years of vaccine research have produced fewhopeful candidates.

Because of the general lack of effective, inexpensive chemotherapeuticagents for treating parasitic protozoal diseases that occur in thedeveloping world, new chemotherapeutic agents are needed. For example,it is estimated that approximately 1.5 -2 million new cases ofleishmaniasis occur each year due to infection by various Leishmaniaspecies.¹ Pentavalent antimonial drugs are the first line treatment forleishmaniasis in most affected areas, with amphotericin B andpentamidine being used as alternatives.² These agents must beadministered by injection over several days to weeks, increasing thecost and inconvenience of the drugs. Resistance to antimonials hasbecome a severe problem,³ and treatment with amphotericin B andpentamidine is frequently complicated by the occurrence of toxic sideeffects.² Clearly, improved chemotherapeutics are needed against thisdisease, as well as the other parasitic diseases.

SUMMARY OF THE INVENTION

The present invention provides a new class of dinitroaniline sulfonamidepharmaceutical compounds useful for the treatment of diseases caused byparasitic protozoa. The invention further provides pharmaceuticalcompositions made from the compounds of the present invention. Theinvention further provides methods of treating subjects having diseasescaused by parasitic protozoa by administering a therapeuticallyeffective amount of a compound of the present invention, or apharmaceutically acceptable salt or ester thereof, to the subject. Thecompounds and methods of the present invention are particularly usefulin the treatment of subjects having leishmanaisis.

The dinitroaniline compounds of the present invention are those ofFormula I:

wherein R₁ and R₂ are selected from the group consisting of H and C₁-C₁₀alkyl, provided that R₁ and R₂ are not both n-propyl; or R₁ and R₂ canform a 5 or 6 membered ring containing C, N, and O, such as morpholinoor pyrrolindino. Preferably R₁ and R₂ are methyl, ethyl, propyl, orbutyl. R₃ is a sulfonamide, which is optionally substituted with C₁-C₁₀alkyl, aryl, alkylaryl, alkoxyaryl, or haloaryl.

A subclass of compounds of special interest are those of Formula II:

wherein R₄ and R₅ are the same and selected from the group consisting ofbutyl, pentyl, and hexyl, or R₄ and R₅ form a pyrrolidine ring; and R₆is selected from the group consisting of H, C₁-C₁₀ alkyl, aryl,alkylaryl, alkoxyaryl, and haloaryl.

Another subclass of interest are the compounds of formula III:

wherein n is 1, 2, 3, or 4 and Y is selected from the group consistingof NH₂, C₆NH, C₆H₄(OCH₃)NH, C₆H₄(CH₃)NH, C₆H₄ClNH, provided that whenn=2 Y is not NH₂. Preferred compounds of formula III include:4(dibutylamino)-3,5-dinitrobenzenesulfonamide;N1-Phenyl-3,5dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methyl)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-Phenyl-3,5-dinitro-N4,N4-di-n-ethylsulfonilamide; andN1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide. Especiallypreferred compounds for the treatment of L. donovani are:N1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide; andN1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide. Especiallypreferred compounds for the treatment of T. b. brucei are:N1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-ni-propylsulfonilamide;N1-(3-Methyl)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-Phenyl-3,5-dinitro-N4,N4-di-n-ethylsulfonilamide; andN1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide.

It is preferred that the compounds of Formulae I, II, and III bind tothe tubulin of the parasitic protozoa. It is further preferred that thecompounds of Formulae I, II, and III preferentially bind to the tubulinof parasitic protozoa rather than mammalian tubulin. In accordance withthe present invention, it is especially preferred that the compounds ofFormulae I, II, and III bind to the tubulin of parasitic protozoa withgreater specificity than oryzalin.

The present invention also relates to pharmaceutical compositionscontaining the antileishmanial dinitroaniline sulfonamide compounds ofthe current invention in a pharmaceutically acceptable carrier. Thepresent invention further relates to pharmaceutically acceptablederivatives of the antileishmanial dinitroaniline sulfonamide compounds.

The present invention further relates to methods of using the compoundsto treat diseases caused by parasitic protozoa, specificallyleishmaniasis, including visceral leishmaniasis, mucocutaneousleishmaniasis, and cutaneous leishmaniasis, caused by protozoa of theLeishmania genus, such as Leishmania donovani. The invention furtherrelates to methods of treating other diseases caused by parasiticprotozoa, including Chagas disease; human African trypanosomiasis, alsoknown as African sleeping sickness; animal trypanosomiasis; and malaria.These diseases are caused by the protozoa Trypanosoma cruzi Trypanosomabrucie gambiense, Trypanosoma brucie rhodesiense, Plasmodium falciparum,Plasmodium vivax, Plasmodium ovali, and Plasmodium malariae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the structures of trifluralin, chloralin, and oryzalin.

FIG. 2 is an analysis of DNA content in L. donovani by treated withcompound 20 by flow cytometry. After 48 h treatments with 1% DMSO(Control) and 10 μM compound 20, parasites were fixed, stained withpropidium iodide, and analyzed by flow cytometry.

FIG. 3 shows the reaction scheme for preparation of compounds 3 and13-21.

FIG. 4 shows the reaction scheme for preparation of compound 25.

FIG. 5 shows the reaction scheme for preparation of compounds 27-29.

DETAILED DESCRIPTION OF THE INVENTION

In the early 1990, reports that the commercial herbicide trifluralin (1,FIG. 1), a dinitroaniline that binds to plant but not animal tubulin,⁴possessed selective antileishmanial activity were therefore cause foroptimism. Trifluralin inhibited the proliferation of Leishmaniaamastigotes in macrophages, and radiolabeled trifluralin was shown tobind better to partially purified leishmanial tubulin than to rat braintubulin.⁵ Trifluralin has also been reported to possess activity inanimal models of leishmaniasis.⁶ Unfortunately, the story oftrifluralin's antiparasitic potential has become more confusing withtime. The synthetic precursor of trifluralin, chloralin (2, FIG. 1), isoften present as an impurity in commercial trifluralin preparations, andchloralin was much more toxic to Leishmania in vitro than trifluralin.⁷We subsequently purified leishmanial tubulin in our own laboratory andfound that chloralin, in addition to being a much more effective invitro antileishmanial agent than trifluralin, was an inhibitor ofleishmanial tubulin polymerization while trifluralin was not.⁸ Otherreports describing the effects of trifluralin and related compounds onprotozoan parasites have appeared,^(9,10) but the effects of thesecompounds on parasite tubulin were not examined Trifluralin alsopresents serious technical problems due to its low solubility.⁴ It istherefore difficult to determine whether 1 is a useful antileishmaniallead compound that targets protozoal tubulin from the studies describedabove.

The antimitotic effects of the related dinitroaniline herbicide oryzalin(3, FIG. 1) on Leishmania have also been described.¹¹ Oryzalin, whichcontains a sulfonamide group in place of the trifluoromethylfunctionality present in trifluralin, possesses approximately tenfoldgreater aqueous solubility than trifluralin.⁴ We obtained a commercialsample of oryzalin and found that it did indeed inhibit the assembly ofpurified leishmanial tubulin and was moderately toxic to Leishmaniaparasites in vitro (K. Werbovetz, unpublished results). On the basis ofthis positive result, we decided to employ oryzalin as a lead compoundto explore the structure-activity relationship of dinitroanilinesagainst leishmanial tubulin and against Leishmania parasites in vitro.

The present invention provides compounds useful for treating diseasescaused by parasitic protozoa specifically Leishmania donovani, and alsoincluding Trypanosoma cruzi, Trypanosoma brucie gambiense, Trypanosomabrucie rhodesiense, Plasmodium falciparum, Plasmodium vivax, Plasmodiumovali, and Plasmodium malariae. The methods of the present inventioncomprise treating a subject affected with a diseased caused by aparasitic protozoan with a therapeutically effective amount of acompound of the present invention or a derivative or pharmaceuticallyacceptable salt or ester thereof.

SYNTHESIS AND EXAMPLES

Synthesis of Compounds 13-21 We first synthesized oryzalin and thirteennew dinitroaniline compounds that differ from 3 in one region of themolecule. Target sulfonamides 3 and 13-21 were prepared as shown inScheme 1. The key intermediate 4-chloro-3,5-dinitrobenzenesulfonic acid4¹² was converted to amines 5-12 by heating 4 to reflux with thecorresponding amine (for dipropyl 5, propyl 6 and diethylamine 7derivatives) or by treating 4 with a methanol solution containing thedesired amine (for dibutyl 8, dipentyl 9, and dihexylamine 10derivatives). The morpholino (11) and pyrrolidino (12) derivatives wereprepared by stifling compound 4 in a methanolic solution containingeither morpholine or pyrrolidine. Sulfonic acids 5-12 were thenconverted to the corresponding sulfonyl chloride derivatives (which werenot isolated due to their unstable nature) by PCl₅/CH₂Cl₂ treatment. Thesulfonyl chlorides were then transformed to sulfonamide derivatives bytreatment with either ammonia in MeOH (3, 13-19), aniline (20), orpropylamine (21).

Synthesis of Compound 25 Mononitro derivative 25 was prepared asindicated in Scheme 2. 1-Chloro-2-nitrobenzene 22 was converted to 23 byheating at 120° C. in chlorosulfonic acid, which was then transformed tothe sulfonamide 24 at −5° C. by reaction with ammonia in dioxane/ethylacetate. Sulfonamide 24 was then converted to 25 by treatment withdipropylamine at reflux.

Synthesis of Compounds 27-29 Cyano, amide and amidoxime compounds 27-29were prepared according to Scheme 3. Commercially available4-chloro-3,5-dinitrobenzonitrile 26 was converted to the correspondingdipropylamine derivative 27 by a modified Ullmann condensation usingdipropylamine under reflux conditions to achieve nearly quantitativeyield. Benzonitrile 27 was converted to amide 28 by hydrolysis withconc. H₂SO₄ at 50° C. The amidoxime derivative 29 was prepared byheating 27 with NH₂OH.HCl/Na₂CO₃ in a solution of ethanol/water atreflux¹³. Target compounds 3, 13-20, 25, and 27-29 were purified bysilica gel column chromatography and crystallization. These moleculespossessed satisfactory ¹H NMR and mass spectra and did not containdetectable traces of electrophilic precursors.

Example 1

Inhibition of Leishmanial Tubulin Assembly for Compounds 3, 13-21, 25,27-29

With the desired compounds in hand, we then examined oryzalin andseveral new dinitroaniline compounds for their ability to block thegrowth of Leishmania donovani parasites and to inhibit the assembly ofpurified leishmanial tubulin by methods described previously in Havens,C.; Bryant, N.; Asher, L.; Lamoreaux, L.; Perfetto, S.; Brendle, J.;Werbovetz, K., “Cellular effects of leishmanial tubulin inhibitors on L.donovani,” Mol. Biochem. Parasitol. 2000, 110, 223-236, incorporatedherein by reference.¹⁴

Briefly, the tubulin assembly assays are performed as follows. Assemblyreactions were performed in 96-well half-area microplates (Costar) in afinal volume of 50 ∝L. For both leishmanial and brain tubulin, reactionscontained final concentrations of 1.5 mg/mL (15 ∝M) tubulin, 0.1 mMPIPES (pH 6.9), 1 mM EGTA, 1 mM MgCl₂, 10% (v/v) DMSO, and 1 mM GTP withand without compounds of interest Components of the reaction mixtureswere added to the microplate on ice, then assembly was initiated by thesimultaneous addition of a GTP solution to all sample wells with amultichannel pipet. The change in turbidity was measured at 351 nm usingthe SpectraMax Plus microplate reader immediately after GTP addition at30° C. (for leishmanial tubulin) or 37° C. (for brain tubulin). Toensure maximal solubility of GB-II-5 at a concentration of 40 ∝M,compounds were added to reactions containing brain tubulin in 5 ∝Lvolumes containing 100% DMSO, and assembly was initiated by adding 10 ∝Lof 5 mM GTP in water. The assembly of leishmanial tubulin occurred toorapidly for accurate measurement under the conditions described forbrain tubulin. Since lower concentrations of 20 affect leishmanialtubulin and solubility was thus not as much of a concern, compounds wereadded to reactions containing parasite tubulin in 5 ∝L volumescontaining 50% DMSO, then assembly was initiated by adding 10 ∝L of 5 mMGTP in 25% DMSO.

Moderate antiparasitic and antitublin activity was observed with 3;mid-micromolar concentrations of this compound inhibited parasite growthand 20 μM concentrations of 3 inhibited the assembly of leishmanialtubulin by about 50% (Table 1). Compound 13, in which the dipropylsubstitution present at the N4 position of the sulfanilamide core of 3is replaced by a propyl group, displays similar antiparasitic activityand activity against the target protein compared to 3. When the alkylchain length was shortened from three to two carbons in compound 14,antiparasitic activity and potency against the target protein were againcomparable with 3. Interestingly, an increase in the length of thedialkylamine chain present in 3 dramatically improved antileishmanialactivity. Compounds 15-17 were 2.5- to 5.5-fold more active against thepromastigote stage of the parasite and 3.6- to 8.1-fold more activeagainst L. donovani axenic amastigotes than 3, while 15 and 16 possesssuperior activity compared to 3 against the putative target protein.Restricting the conformation of the alkylamine chain, as in themorpholino compound 18 and the pyrrolidino compound 19, decreasedactivity against Leishmania parasites. A difference between 18 and 19was noted in antitubulin activity, as the pyrrolidino compound 19 wassimilar to oryzalin against tubulin but the morpholino compound 18 wasinactive against the protein. TABLE 1 Activity of Oryzalin and Newdinitroaniline compounds 13-29 Against L. donovani and AgainstLeishmanial Tubulin in vitro % Inhibition of IC₅₀ vs. Leishmanial L.donovani Tubulin Assembly IC₅₀ vs. L. donovani amastigotes at 20 μMCompound promastigotes (μM)^(a) (μM)^(a) Compound^(b) Pentamidine 1.61 ±0.22 2.00 ± 0.06 NT^(c) 3 (oryzalin) 44.1 ± 9.2  72.5 ± 23.9 54 ± 14 1367.0 ± 18.4 NT 43 ± 19 14 69.3 ± 23.0 NT 58 ± 22 15 17.8 ± 2.6  20.1 ±0.2  89 ± 3  16 8.02 ± 0.42 9.0 ± 0.7 95 ± 5  17 12.2 ± 0.0  11.7 ± 0.7 48 ± 19 18 >100 NT 21 ± 1  19 97.1 ± 53.0 NT 57 ± 18 20 14.7 ± 1.0  5.41± 0.89 108 ± 7  21 56.0 ± 2.3  56.9 ± 3.4  97 ± 1  25 89.8 ± 15.3 NT 19± 29 27 >100 NT 25 ± 17 28 76.4 ± 20.0 NT 34 ± 12 29 >100 NT 35 ± 7 ^(a)Mean ± standard deviation of at least two independent measurements^(b)Mean ± standard deviation of at least one duplicate measurement. Thestandard deviation of the control samples in these experiments was 27.6%^(c)NT—Not tested

Another striking result was obtained with compounds 20 and 21, bothsubstituted at the N1 position of the sulfanilamide core, in that thesetwo compounds are much more potent inhibitors of leishmanial tubulinassembly than 3. Compound 20 is 3.0-fold more active againstpromastigotes and 13.4-fold more active against amastigotes thanoryzalin 3, approaching the in vitro antiparasitic activity of theclinical antileishmanial agent pentamidine against the form of theparasite present in the mammalian host. Despite its improved activityagainst leishmanial tubulin, compound 21 is similar to 3 inantiparasitic activity. Other substitutions diminished the activity ofthe compounds. Removal of a single nitro group, as in 25, decreases thepotency against cultured parasites and leishmanial tubulin. Replacementof the sulfonamide group with a cyano group, an amide functionality, oran amidoxime group as in 27-29 diminishes activity against L. donovaniand leishmanial tubulin as well.

Our study clearly demonstrates that the pure dinitroanilines possessantileishmanial and antitubulin activity. While other reports showedthat several different dinitroaniline compounds possessed antiparasiticactivity,^(9,15) evidence linking dinitroanilines with tubulininhibition in parasites was less clear. In addition to chloralin 2,other small aromatic electrophiles are known to inhibit tubulin assemblyin general^(16,17) and leishmanial tubulin assembly in particular.¹⁴This report shows that several non-electrophilic nitroaniline compoundsdisplay potent effects against Leishmania parasites and against purifiedleishmanial tubulin, renewing hopes that such compounds could be leadsfor the discovery of new antiparasitic agents.

It is also clear that several synthetic, non-electrophilicdinitroaniline compounds are superior to 3 in their activity againstleishmanial tubulin and cultured Leishmania parasites. Compounds 15-17and 20 are significantly more potent against parasites than leadcompound 3, with 20 being 13.4-fold more active than 3 againstamastigote-stage L. donovani, and 15, 16, 20 and 21 are more potent than3 in blocking leishmanial tubulin assembly. These data are consistentwith the hypothesis that tubulin is the target of the dinitroanilines inLeishmania. Although an exact correlation was not observed betweenantiparasitic activity and the inhibition of leishmanial tubulinassembly, all of the compounds that are superior to oryzalin inantiparasitic activity are at least as potent as 3 in blocking parasitetubulin assembly in vitro. Thus inhibition of leishmanial tubulinpolymerization appears to be a necessary but not sufficient property formembers of this class of compounds to possess strong antileishmanialactivity. An important factor that may help explain the lack of a directcorrelation between antitubulin and antileishmanial activity of thesecompounds is penetration of the molecules into the parasite. Compounds15-17 have longer alkyl chains than 3 and would thus be expected to bemore hydrophobic than the parent compound, facilitating passage ofcompounds 15-17 across the parasite cell membrane. The difference inactivity between 20 and 21 is harder to rationalize. Although bothcompounds are more potent inhibitors of leishmanial tubulin assemblythan oryzalin, N1-phenyl sulfanilamide 20 is much more effective than 3at blocking parasite growth, while N1-alkyl sulfanilamide 21 and 3possess similar antiparasitic activity. Poor accumulation of 21 withinthe parasite may also explain these data.

Example 2

Cellular Effects of the New Dinitroaniline Compounds on Leishmania

We have verified that our new agents act through a microtubule mechanismin the parasites with the aid of flow cytometry and fluorescencemicroscopy.

Flow cytometry and fluorescence cell sorting. Parasites or J774macrophages were incubated in T25 flasks (3-5 mL final volume) in theappropriate media described earlier for 24-48 h. Cultures contained 1%DMSO (v/v) in the presence or absence of test compounds. Cells werecounted using a hemacytometer, then were centrifuged (1000×·g forparasites, 200×·g for macrophages) at 4° C. for 10 min (parasites) or 5min (macrophages). Cells were resuspended in 150 μL of PBS (0.01 Mphosphate, 0.137 M NaCl, and 2.7 mM KCl), then 350 μL of ice-coldmethanol was added and the samples were fixed at −20° C. for 2-3 h.Cells were centrifuged as before and resuspended in PBS containing 0.1%TX-100, 5 μg/mL RNase A, and 10 μg/mL propidium iodide for 20-30 min.Centrifugation was repeated, then cells were resuspended in PBS to afinal concentration of 5×10⁵-10⁶/mL and stored at 4° C. until analysis.Fluorescence cell sorting was conducted on a Beckman Coulter Elite flowcytometer.

Fluorescence microscopy. L. donovani promastigotes were incubated withor without test compounds for two days and centrifuged as describedabove. Parasites were resuspended in PBS at a concentration of 2×10⁷cells/mL, then 10⁶ parasites were applied to a microscope slidepreviously coated with 0.1 mg/mL poly-L-lysine. Parasites were allowedto adhere for 10 min, then the cells were fixed in methanol for 5 min,rinsed with PBS, then placed in 0.1% TX-100 in PBS for 5 min. Slideswere then washed with PBS, and were incubated with a 1:100-1:1000dilution of a FITC-conjugated anti α-tubulin antibody (clone DM 1A) for1 h at room temperature. After washing the slides 3×· with PBS for 5 mineach, 4′,6-diamidino-2-phenylindole (DAPI) was applied to slides at aconcentration of 10 μg/mL. Slides were washed again in PBS, thencoverslips were mounted using Vectashield (Vector Laboratories,Burlingame, Calif.) in preparation for fluorescence microscopy using aNikon Labophot-2 microscope equipped with a Nikon FX-35DX camera.

Flow cytometry studies indicated that a 48 hour treatment of L. donovanipromastigotes with 10 μM compound 15 caused parasites to accumulate inthe G₂M phase of the cell cycle, consistent with an effect onmicrotubules. Compound 15 at 10 μM was more potent in this flowcytometry assay than 50 μM concentrations of oryzalin. Even moredramatic effects were observed with compound 20, as treatment ofpromastigotes with 10 μM concentrations of this compound for 48 hourscaused the appearance of parasites containing approximately 4×, 8×, and16× more DNA than those observed in the G₁ phase of control samples(FIG. 2, note log scale). In this experiment, parasites in controlcultures doubled approximately three times over the course of 48 hours,while compound 20-treated parasites remained at the same cell density.These results suggest that the compound 20-treated parasites failedmitosis after S-phase due to microtubule inhibition, but then continuedthrough the cell cycle only to fail mitosis on one, two, or threeadditional occasions. Brightfield microscopy revealed aberrant formsthat resembled cultures treated with the potent antimitotic naturalproduct ansamitocin P3, and DAPI staining of these cultures showed thatthese parasites frequently contained multiple kinetoplasts as weobserved previously in promastigotes treated with other knownantimitotics.¹⁴

Example 3

Selectivity of the Near Dinitroaniline Compounds

Since we are ultimately interested in developing antiparasitic agentsthat will be nontoxic to the host, selectivity is a critical parameterregarding these compounds. We have determined the selectivity of ourcompounds by using tubulin fluorescence quenching experiments.

Measurement of dissociation constants by fluorescence quenching. Bindingof dinitroanilines to tubulin was measured by the resulting quenching ofintrinsic tubulin tryptophan fluorescence, a method that has proveduseful in protein binding studies of many compounds (23). The strong300-350 nm absorbance of these compounds makes them good acceptors ofresonance energy transfer from the photoexcited tryptophan residues,resulting in reduction of tryptophan fluorescence. Samples of tubulinwith increasing concentrations of test compound were excited at 290 nm,and tryptophan fluorescence measured between 310 and 340 nm. Spectrawere recorded using an ISS PC1 photon counting spectrofluorometer (ISS,Champaign, Ill.) with excitation and emission slits of 2 and 1 mm,respectively. Sample fluorescence was corrected for the smallphotobleaching by measuring a tubulin sample with additions of DMSO.Inner filter effects were corrected using a sample ofN-acetyltryptophanamide, and additions of the test compound. Thecorrected fluorescence decrements as a function of test compoundconcentration were fitted to a single site binding model using thenonlinear fitting routines of Prism software (Graph Pad Software, SanDiego, Calif.).

The results confirm our earlier conclusions that several of our newdinitroaniline compounds are better ligands for leishmanial tubulin thanthe lead compound (Table 2). These data are also in agreement withresults indicating that oryzalin is a mediocre ligand for leishmanialtubulin, and provide the first quantitative measure of the selectivityof oryzalin for this protein. With the exception of compound 15, all ofour compounds are more selective for leishmanial tubulin than mammaliantubulin, and the N1-substituted compounds, compound 20 and compound 21are more selective than oryzalin for leishmanial tubulin. TABLE 2Dissociation Constants for New dinitroaniline compounds AgainstLeishmanial and Rat Brain Tubulin K_(d) (μM) vs. Leishmanial K_(d) (μM)vs. Brain Selectivity Compound Tubulin Tubulin Index Oryzalin, 3 170  24 7.1 20  57    5 11.4 21 (770)*  29 26.6 15  19   21 0.9 16  54   124.5 17  39    8 4.9 ColchAC  5  (1300) 0.004

Additional compounds were prepared as follows: TABLE 1 AdditionalCompounds

Compound n Y 3 2 NH₂ (oryzalin) 15, above 3 NH₂ 20, above 2

30 2

31 1

32 3

Comparative Examples 33 2 NHCH₃ 34 2 N(CH₃)₂ 35 2 NHCH₂CH₃ 36 2N(CH₂CH₃)HD 2 37 2 NH(CH₂)₂CH₃ 38 2 NHCH(CH₃)₂ 39 2

40 2

41 2

42 2

43 2

44 2

45 2

46 2

Example 4

N1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (20) wassynthesized by the reaction of the sulfonyl chloride and 547 μL (6 mmol)of aniline in 20 mL pyridine. The product was isolated by chromatography(hexane/EtOAc (3:1)) and crystallized by dichloromethane/hexane toafford a yellow crystalline solid; yield 342 mg, 81%, mp=120° C.; ¹HNMR³ ¹³C NMR (DMSO-d₆)³. MS (FAB) m/z Calcd for C₁₈H₂₂H₄O₆S(M+Na)⁺445.1158, Measured (M+Na)⁺445.1164. Anal. (C₁₈H₂₂N₄O₆S) C, H, N.

Example 5

N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (30) wassynthesized by the reaction of the sulfonyl chloride and 369 mg (3 mmol)of 4-methoxyaniline in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (6:1)) and crystallized usingdichloromethane/hexane to obtain a yellow crystalline solid; yield14.5%; mp.=133 ° C.; ¹H NMR (CDCl₃) δ 0.86 (t, 6H, J=7.33 Hz), 1.58 (m,4H), 2.93 (t, 4H, J=7.40 Hz), 3.78 (s, 3H), 6.49 (bs, 1H), 6.75 (m, 3H),7.21 (m, 1H), 8.12 (s, 2H); ¹³C NMR (CDCl₃) δ 11.00, 20.55, 53.89,55.39, 107.45, 111.70, 113.62, 128.73, 128.89, 130.38, 136.51, 141.72,144.26, 160.46. MS m/z 451.1 (M−H)⁻. Anal. Calcd. For C₁₉H₂₄N₄O₇S C, H,N.

Example 6

N1-Phenyl-3,5-dinitro-N4,N4-di-n-ethylsulfonilamide (31).

The sulfonyl chloride derivative was prepared from the sulfonic acid andPCl₅. It was then treated with 547 μL (6 mmol) of aniline in 20 mLpyridine. The product was isolated by chromatography (hexane/EtOAc(6:1)) and crystallized using dichloromethane/hexane to afford a yellowcrystalline solid; yield 262 mg, 67%; mp=157° C. ¹H NMR (DMSO-d₆) δ 0.99(t, 6H, J=7.00 Hz), 2.99 (q, 4H, J=7.00 Hz), 7.17 (m, 5H), 8.28 (s, 2H),10.45 (bs, 1H); ¹³C NMR (DMSO-d₆) δ 12.54, 45.60, 120.82, 124.95,127.92, 129.43, 130.69, 136.73, 140.63, 144.96. MS m/z 392.9 (M−H)⁻.Anal. (C₁₆H₁₈N₄O₆S) C, H, N.

Example 7

N1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide (32).

The sulfonyl chloride derivative was prepared as before from thesulfonic acid and PCl₅. It was then treated with 547 μL (6 mmol) ofaniline in 20 mL pyridine. The product was isolated by chromatography(hexane/EtOAc (6:1)) and crystallized by dichloromethane/hexane toafford a yellow crystalline solid. Yield 286 mg, 64%; mp=156° C. ¹H NMR(CDCl₃) δ 0.87 (t, 6H, J=7.50 Hz), 1.28 (m, 4H), 1.55 (m, 4H), 2.96 (t,4H, J=7.50 Hz), 6.57 (bs, 1H), 7.25 (m, 5H), 8.10 (s, 2H). ¹³C NMR(CDCl₃) δ 13.66, 19.85, 29.35, 52.07, 122.18, 126.52, 128.61, 129.09,129.78, 135.24, 141.66, 144.42. m/z Calcd For C₂₀H₂₆N₄O₆S (M−H)⁻ 449.0.Anal. (C₂₀H₂₆N₄O₆S) C, H, N.

Comparative Example A

N1-Methyl-3,5-dinitro-N4,N4di-n-propylsulfonilamide (33) was synthesizedby the reaction of the sulfonyl chloride and 10 mL of 2 M methylamine inTHF. The product was isolated by chromatography using hexane/EtOAc 2:1as eluent and crystallized using dichloromethane/hexane to obtain yellowcrystals; yield 88 mg, 97%; mp=148 ° C.; ¹H NMR (CDCl₃) δ 0.86 (t, 6H,J=7.25 Hz), 1.62 (m, 4H), 2.76 (d, 3H, J=5.25 Hz), 2.97 (m, 4H), 4.38(bq, 1H), 8.25 (s, 2H); ¹³C NMR (CDCl₃) δ 11.06, 20.63, 29.31, 53.94,128.47, 129.55, 141.48, 144.61. MS m/z 361.00 (M+H)⁺. Anal.(C₁₃H₂₀N₄O₆S) C, H, N.

Comparative Example B

N1,M1-Dimethyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (34) wassynthesized by the reaction of the sulfonyl chloride and 10 mL of 2 Mdimethylamine in THF. The product was isolated by chromatography(hexane/EtOAc (2:1) and crystallized using dichloromethane/hexane toobtain yellow crystals; yield 85 mg, 91%; mp=138° C.; ¹H NMR (CDCl₃) δ0.89 (t, 6H, J=7.25 Hz), 1.63 (m, 4H), 2.81 (s, 6H), 2.98 (m, 4H), 8.15(s, 2H); ¹³C NMR (CDCl₃) δ 6 11.05, 20.62, 37.76, 53.89, 126.88, 128.64,141.47, 144.65. MS m/z 397.00 (M+Na)⁺. Anal. (C₁₄H₂₂N₄O₆S) C, H, N.

Comparative Example C

N1-Ethyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (35) was synthesizedby the reaction of the sulfonyl chloride and 10 mL of 2 M ethylamine inTHF. The product was isolated by chromatography (hexane:EtOAc=3:1) andcrystallized using dichloromethane/hexane to obtain yellow crystals;yield 78 mg, 83%; mp=131° C.; ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=7.25 Hz),1.20 (t, 3H, J=7.25 Hz), 1.62 (m, 4H), 3.08 (m, 6H), 4.44 (bt, 1H), 8.24(s, 2H); ¹³C NMR (CDCl₃) δ 11.06, 15.13, 20.63, 38.41, 53.94, 128.32,130.64, 141.39, 144.61. MS m/z 372.9 (M−H)⁻. Anal. (C₁₄H₂₂N₄O₆S) C, H,N.

Comparative Example D

N1,N1-Diethyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (36) wassynthesized by the reaction of the sulfonyl chloride and 10 mL (0.79mol) of diethylamine. The product was isolated by chromatography(hexane:EtOAc (6:1)) and crystallized using dichloromethane/hexane toobtain yellow crystals; yield 73 mg, 73%; mp=102° C.; ¹H NMR (CDCl₃) δ0.88 (m, 6H), 1.20 (t, 6H, J=7.25 Hz), 1.62 (m, 4H), 2.97 (m, 4H), 3.28(q, 4H, J=7.00 Hz), 8.18 (s, 2H); ¹³C NMR (CDCl₃) δ 11.05, 14.27, 20.63,42.38, 53.92, 128.03, 131.35, 141.07, 144.73. MS m/z 403.1 (M+H)⁺. Anal.(C₁₆H₂₆N₄O₆S) C, H, N.

Comparative Example E

N1-Propyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (37) wassynthesized by the reaction of the sulfonyl chloride and 10 mL (0.12mol) of propylamine. The product was isolated by chromatography(hexane:EtOAc (3:1)) and crystallized using dichloromethane/hexane toobtain yellow crystals; yield 86 mg, 88%; mp=114° C.; ¹H NMR (DMSO-d₆) δ0.82 (m, 9H), 1.55 (m, 6H), 2.97 (m, 6H), 7.88 (bs, 1H), 8.37 (s, 2H);¹³C NMR (DMSO-d₆) δ 11.24, 11.38, 20.74, 22.77, 44.64, 53.71, 128.27,132.28, 140.59, 145.11. MS m/z 389.3 (M+H)⁺. Anal. (C₁₅H₂₄N₄O₆S) C, H,N.

Comparative Example F

N1-Isopropyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (38) wassynthesized by the reaction of the sulfonyl chloride and 10 mL (0.12mol) of isopropylamine. The product was isolated by chromatography(hexane/EtOAc (6:1)) and crystallized using dichloromethane/hexane togive a yellow crystalline solid; yield 77 mg, 79%; mp=150° C.; ¹H NMR(CDCl₃) δ 0.88 (t, 6H, J=7.25 Hz), 1.27 (d, 6H, J=7.25 Hz), 1.63 (m,4H), 2.97 (m, 4H), 3.57 (m, 1H), 4.41 (bs, 1H), 8.25 (s, 2H); ¹³C NMR(CDCl₃) δ 11.11, 20.70, 23.85, 46.68, 54.07, 128.22, 131.99, 141.33,144.71. MS m/z 389.0 (M+H)⁺. Anal. (C₁₅H₂₄N₄O₆S) C, H, N.

Comparative Example G

N1-(4-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-Propylsulfonilamide (39) wassynthesized by the reaction of the sulfonyl chloride and 369 mg (3 mmol)of 4-methoxyaniline in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (6:1)) and crystallized usingdichloromethane/hexane to obtain a yellow crystalline solid; yield 221mg, 69%; mp=121° C.; ¹H NMR (CDCl₃) δ 0.86 (t, 6H, J=7.25 Hz), 1.61 (m,4H), 2.91 (m, 4H), 3.79 (s, 3H), 6.58 (bs, 1H), 6.83 (dd, 2H, J=2.00,7.00Hz), 7.30 (dd, 2H, J=2.00, 7.00 Hz), 8.04 (d, 2H, J=0.75 Hz); ¹³CNMR (CDCl₃) δ 11.08, 20.65, 53.99, 55.49, 114.85, 125.92, 127.42,128.60, 129.31, 141.53, 144.40, 158.71. MS (FAB) m/z Calcd forC₁₉H₂₄N₄O₇S (M+Na)⁺ 475.1263, Measured (M+Na)⁺ 475.1275. Anal.(C₁₉H₂₄N₄O₇S) C, H, N.

Comparative Example H

N1-(4Methyl)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (40) wassynthesized by the reaction of the sulfonyl chloride and 321 mg (3 mmol)of 4-methylaniline in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (6:1)) and crystallized usingdichloromethane/hexane to obtain yellow crystals; yield 338 mg, 77%;mp=107° C.; ¹H NMR (CDCl₃) δ 0.85 (t, 6H, J=7.25 Hz), 1.60 (m, 4H), 2.31(s, 3H), 2.91 (t, 4H, J=7.75 Hz), 6.77 (bs, 1H), 7.10 (d, 2H, J=8.25Hz), 7.20 (d, 2H, J=8.25 Hz), 8.10 (d, 2H, J=0.75 Hz)); ¹³C NMR (CDCl₃)δ 11.08, 20.65, 20.85, 53.99, 122.87, 128.61, 129.33, 130.27, 132.44,136.76, 141.61, 144.39. MS (FAB) m/z Calcd for C₁₉H₂₄N₄O₆S(M+Na)⁺459.1314, Measured (M+Na)⁺459.1316. Anal. (C₁₆H₂₄N₄O₆S) C, H, N.

Comparative Example I

N1-(4-Chloro)phenyl-3,5dinitro-N4,N4-di-n-propylsulfonilamide (41) wassynthesized by the reaction of the sulfonyl chloride and 351 mg (3 mmol)of 4-chloroaniline in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (6:1)) and crystallized bydichloromethane/hexane to obtain yellow crystals; yield 258 mg, 58%;mp=130° C.; ¹H NMR (CDC₃) δ 0.86 (t, 6H, J=7.50 Hz), 1.61 (m, 4H), 2.94(m, 4H), 6.95 (bs, 1H), 7.09 (d, 2H, J=7.25 Hz), 7.30 (d, 2H, J=7.25Hz), 8.14 (d, 2H, J=0.15 Hz); ¹³C NMR (CDCl₃) δ 11.09, 20.65, 54.03,123.37, 128.65, 128.77, 129.92, 132.21, 133.75, 141.85, 144.38. MS (FAB)m/z Calcd for C₁₈H₂₁ClN₄O₆S (M+Na)⁺ 479.0768, Measured (M+Na)+ 479.0767.Anal. (C₁₈H₂₁ClN₄O₆S) C, H, N.

Comparative Example J

N1-(3-Methyl)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (42) wassynthesized by the reaction of the sulfonyl chloride and 321 mg (3 mmol)of 4-methylaniline in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (6:1)) and crystallized usingdichloromethane/hexane to obtain a yellow crystalline solid; yield15.2%; mp.=103° C.; ¹H NMR (CDCl₃) δ 0.86 (m, 6H), 1.57 (m, 4H), 2.31(s, 3H), 2.93 (t, 4H, J=7.44 Hz), 6.67 (bs, 1H), 6.90 (m, 2H, J=7.25Hz), 7.03 (d, 1H, J=0.67 Hz), 7.19 (m, 1H), 8.11 (s, 2H); ¹³C NMR(CDCl₃) δ 11.03, 20.58, 21.28, 53.93, 119.08, 122.79, 127.25, 128.56,129.16, 129.47, 135.04, 139.92, 141.57, 144.28. MS m/z 435.1 [M−H]⁻.Anal. (C₁₆H₂₄N₄O₆S) C, H, N.

Comparative Example K

N1-(3-Chloro)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (43) wassynthesized by the reaction of the sulfonyl chloride and 351 mg (3 mmol)of 4-chloroaniline in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (6:1)) and crystallized bydichloromethane/hexane to obtain yellow crystals; yield 23.4%; mp.=108°C.; ¹H NMR (CDCl₃) δ 0.87 (t, 6H, J=7.25 Hz), 1.59 (m, 4H), 2.95 (t, 4H,J=7.43 Hz), 6.74 (s, 1H), 7.17 (m, 4H), 8.16 (s, 2H); ¹³C NMR (CDCl₃) δ11.02, 20.57, 53.94, 119.46, 121.67, 126.37, 128.63, 130.73, 135.32,136.44, 141.85, 144.26. MS m/z 455.1 [M−H]⁻. Anal. (C₁₈H₂₁ClN₄O₆S) C, H,N.

Comparative Example L

N1-(3,4-Dichloro)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (44)was synthesized by the reaction of the sulfonyl chloride and 486 mg (3mmol) of 3,4-dichloro aniline in 20 mL pyridine. The product wasisolated by chromatography (hexane/EtOAc (6:1)) and crystallized bydichloromethane/hexane to obtain a yellow crystalline solid; yield 264mg, 54%; mp=111° C.; ¹H NMR (CDCl₃) δ 0.88 (m, 6H), 1.61 (m, 4H), 2.95(m, 4H), 6.84 (bs, 1H), 7.01 (dd, 1H, J=2.50, 8.75 Hz)), 7.23 (d, 1H,J=2.50 Hz), 7.40 (d, 1H, J=8.75 Hz), 8.16 (s, 2H); ¹³C NMR (CDCl₃) δ11.06, 20.63, 54.02, 120.64, 123.27, 128.27, 128.77, 130.16, 131.33,133.61, 134.84, 142.04, 144.34. MS (FAB) m/z Calcd for C₁₈H₂₀ClN₄O₆S(M+Na)⁺ 513.0378, Measured (M+Na)⁺ 513.0388. Anal. (C₁₈H₂₂N₄O₆S) C, H,N.

Comparative Example M

N1-(3,5-Dichloro)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (45)was synthesized by the reaction of the sulfonyl chloride and 486 mg (3mmol) of 3,5dichloro aniline in 20 mL pyridine. The product was isolatedby chromatography (hexane/EtOAc (6:1)) and crystallized bydichloromethane/hexane to obtain yellow crystals; yield 356 mg, 73%;mp=144° C.; ¹H NMR (CDCl₃) δ 0.87 (t, 6H, J=7.50 Hz), 1.63 (m, 4H), 2.95(q, 4H, J=6.63 Hz), 6.69 (bs, 1H), 7.06 (d, 1H, J=0.17 Hz), 7.20 (d, 2H,J=0.17 Hz), 8.17 (d, 2H, J=0.25 Hz); ¹³C NMR (CDCl₃) δ 11.01, 20.58,53.99, 119.28, 126.13, 128.14, 128.64, 136.02, 137.27, 142.02, 144.30.MS m/z 489.1 (M−H)⁻. Anal. (C₁₈H₂₀ClN₄O₆S) C, H, N.

Comparative Example N

N1-(2-Pyridinyl)-3,5-dinitro-N4,N4-di-n-propylsulfonilamide (46) wassynthesized by the reaction of the sulfonyl chloride and 564 mg (6 mmol)of 2-aminopyridine in 20 mL pyridine. The product was isolated bychromatography (hexane/EtOAc (1:1) and crystallized usingdichloromethane/hexane to afford yellow crystals; yield 326 mg, 62%;mp=204° C.; ¹ H NMR (CDCl₃) δ 8 0.86 (t, 6H, J=7.50 Hz), 1.62 (m, 4H),2.94 (m, 4H), 6.90 (m, 1H), 7.47 (d, 1H, J=8.00 Hz), 7.85 (m, 1H), 8.24(m, 1H′), 8.31 (s, 2H), 8.65 (s, 1H); ¹³C NMR (CDCl₃) δ 11.17, 11.25,20.65, 20.75, 53.67, 54.03, 114.02, 116.12, 127.94, 133.61, 138.62,141.11, 144.12, 144.83, 155.69. MS m/z 424.0 (M+H)⁺. Anal. (C₁₈H₂₂N₄O₆S)C, H, N.

The IC₅₀ values for the compounds of Examples 4-7 and ComparativeExamples 8-21 against protozoan parasites and mammalian cells aresummarized below: L. donovani axenic T. b. brucei J774 Compoundamastigotes^(a,b) variant 221^(b,c) macrophages^(a,f) 15 (10) 20.1 ±0.2  2.57 ± 0.70 8.43 ± 0.69 20 (11) 5.03 ± 1.08 0.4 28.3 ± 3.8  30 (15)8.11 ± 3.33 0.5 25.3 ± 3.8  31 (20) 10.6 ± 2.6  1.9 47.7 ± 1.6  32 (21)2.57 ± 0.25 0.1 11.9 ± 0.5   3 (Oryzalin) 64.8 ± 3.6  11.4 ± 0.6  32.9 ±1.6  33 (3) >100 4.6 Not tested 34 (4) >100 5.9 Not tested 35 (5) >1004.0 Not tested 36 (6) 26.7 ± 0.4  10.2 Not tested 37 (7) 54.3 ± 8.5  9.2Not tested 38 (9) 42.8 ± 2.3  2.5 Not tested 39 (12) 31.9 ± 14.6 20.515.2 ± 6.7  40 (13) 32.1 ± 12.7 10.5 10.5 ± 2.0  41 (14) 13.3 ± 3.0  9.59.13 ± 3.81 42 (16) 22.7 ± 0.8  0.92 15.8 ± 2.1  43 (17) 5.47 ± 0.100.88 8.67 ± 0.17 44 (18) 5.03 ± 0.80 6.7 7.19 ± 0.59 45 (19) 3.71 ± 0.913.4 5.12 ± 0.35 46 (22) 59.7 ± 6.2  10.9 44.9 ± 16.3 Pentamidine 2.00 ±0.06 0.0194 ± 0.0069 Not tested Suramin Not tested 0.233 ± 0.050 Nottested

Example 8

IC₅₀ values for Compounds 3, 20, 37, 15, and 17 against severaldifferent protozoan parasites and mammalian cell lines were testedChemicals and biochemicals. Where not otherwise indicated, commercialmaterials were obtained from Sigma. Dinitroaniline sulfonamides weresynthesized as outlined above. Ansamitocin P3 was obtained from the druginventory of the National Cancer Institute.

Determination of the drug susceptibility of parasites and mammaliancells. The susceptibility of Leishmania donovani amastigote-likeparasites (WHO designation: MHOM/SD/62/1S-CL2_(D)) to growth inhibitionby compounds of interest was measured in a three-day assay using thetetrazolium dye-based CellTiter reagent (Promega) (Werbovetz, K.,Brendle, J., and Sackett, D. (1999) Mol. Biochem. Parasitol. 98, 53-65.,and Havens, C., Bryant, N., Asher, L., Lamoreaux, L., Perfetto, S.,Brendle, J., and Werbovetz, K. (2000) Mol. Biochem. Parasitol. 110,223-236). The amastigote medium used in this assay is based on themedium mentioned by Joshi et al. (Joshi, M., Dwyer, D. M., and Nakhasi,H. L. (1993) Mol. Biochem. Parasitol. 58, 345-354). Prior to addition offetal bovine serum to a final concentration of 20%, this amastigotemedium contains 15 mM KCl, 115 mM KH₂PO₄, 10 mM K₂HPO₄, 0.5 mM MgSO₄, 24mM NaHCO₃, a 1-concentration of RPMI-1640 vitamins and amino acids, 2.0mM L-glutamine, 22 mM D-glucose, 100 units/mL penicillin, 100 ∝g/mlstreptomycin, 0.1 mM adenosine, 1 ∝g/mL folate, and 25 mM MES (pH 5.5).Compounds were tested for their activity against bloodstream-formTrypanosoma brucei brucei (MITat 1.2, variant 221) axenically-culturedin HMI-9 medium , H., and Hirumi, K. (1989) J. Parasitol. 75, 985-989.)following the procedure of Ellis et al. (Ellis, J., Fish, W., Sileghem,M., and McOdimba, F. (1993) Vet. Parasitol. 50, 143-149.) with somemodifications. Briefly, 100 μL of late log phase parasites wereincubated in 96-well plates (Costar) at an initial concentration of 10⁵cells/ml with or without test compounds at 37° C. in a humidified 5% CO₂atmosphere for 72 hours. Twenty-five μL of a 5 mg/mL solution of MTT(prepared in phosphate buffered saline and filter sterilized) was thenadded to each well and plates were re-incubated at 37° C. as before for2 h. One hundred ∝L of 20% SDS lysis buffer (prepared in 50% aqueousDMF) was added to each well and plates were incubated as before for anadditional 3-4 h. Optical densities were then measured at 570 nm usingSpectraMax Plus microplate reader. IC₅₀ values, the concentration of thecompound that inhibited cell growth by 50% compared to untreated controlwere determined with the aid of the software program SoftMax Pro(Molecular Devices). This program uses the dose-response equationy=((a−d)/(1+(x/c)_(b)))+d, where x=the drug concentration, y=absorbanceat 490 nm, a=upper asymptote, b=slope, c=IC₅₀ and d=lower asymptote.Assessment of the in vitro sensitivity of T. b. brucei Lab 110 EATRO andP. falciparum strains to compounds was performed by methods described inDonkor et al. (Donkor, I., Assefa, H., Rattendi, D., Lane, S., Vargas,M., Goldberg, B., and Bacchi, C. (2001) Eur. J. Med. Chem. 36, 531-538.)and Guan et al. (Guan, J., Kyle, D., Gerena, L., Zhang, Q., Milhous, W.,and Lin, A. (2002) J. Med. Chem. 45, 2741-2748).

The toxicity of compounds to J774 murine macrophages and PC3 coloncarcinoma cells was measured in a three-day assay using the CellTiterreagent. J774 macrophages in DMEM medium supplemented with 10% heatinactivated fetal calf serum, 2.0 mM L-glutamine, 50 units/mLpenicillin, and 50 μg/mL streptomycin were added to individual wells ofa 96well plate at a concentration of 10₄ cells/L and a total volume of100 μL. Macrophages were allowed to adhere for 24 hours, then the mediumwas removed and replaced with serial dilutions of the test compounds inthe DMEM medium mentioned above without phenol red. After 72 hrincubation with the test compounds at 37° C. in a humidified 5% CO₂incubator, cell viability was determined using the CellTiter reagent byadding 20 μL of assay solution to each well. After a 6-7 h incubation at37° C. to allow for color development, the absorbance of each well at490 nm was measured in a SpectraMax Pro microplate reader (MolecularDevices). The toxicity of compounds to PC3 cells was assessed in thesame manner as for the macrophages, except that the medium used was RPMI1640 supplemented with 10% heat inactivated fetal calf serum, 2.0 mML-glutamine, 50 units/mL penicillin, and 50 ∝g/mL streptomycin. IC₅₀values for the J774 and PC3 assays were determined with the softwareprogram SoftMax Pro as described earlier. TABLE 4 IC₅₀ Values forvarious Compounds (μM) Against Protozoan Parasites and Mammalian CellLines L. donovani T. b. brucei axenic T. b. brucei Lab 110 P. falciparumP. falciparum J774 PC3 Compound amastigotes^(a,b) variant 221^(b,c)EATRO^(d) D6 strain^(e) W2 strain^(e) macrophages^(a) prostate^(a,f) 372.5 ± 23.9 11.4 ± 0.6  5.0 4.0 3.6 32.9 ± 1.6  57.8 ± 10.4 20 5.41 ±0.89 0.412 ± 0.044 0.58 11 13 30.8 ± 1.9  41.3 ± 8.5  37 56.9 ± 3.4 8.06 ± 0.64 3.0 13 >25 21.8 ± 2.1  40.5 ± 8.6  15 20.1 ± 0.2  2.57 ±0.70 1.8 5.1 4.3 8.43 ± 0.69 17.2 ± 2.8  17 11.7 ± 0.7  9.48 ± 1.73 5.610 12 1.91 ± 0.36 11.2 ± 1.3 ^(a)Values represent the mean ± SD of at least two independentexperiments^(b)IC50 values determined by tetrazolium dye assay after three-dayincubation with test compounds. The starting concentration of cells was106 parasites/ml^(c)IC50 values determined by tetrazolium dye assay after three-dayincubation with test compounds. The starting concentration of cells was105 parasites/ml^(d)IC50 values determined by Coulter counter after two-day incubationwith test compounds as described in Donkor et al. (21) The startingconcentration of cells was 105 parasites/ml^(e)IC50 values determined by tritiated hypoxanthine uptake as describedin Guan et al. (22). At the start of the experiment, erythrocytes wereat 0.2% parasitemia and 1% hemotocrit.^(f)IC50 values determined by tetrazolium dye assay after three-dayincubation with test compounds. The starting concentration of cells was104 cells/ml

As used herein, the term “treatment” includes partial or totalinhibition or retardation of the undesirable proliferating parasiteswith minimal inhibition or retardation of normal cells in the vertebratehost. Without limiting the scope of the present invention, a desiredmechanism of treatment is the selective binding of the compounds of thepresent invention with the tubulin of the protozoan with minimal or nobinding to the tubulin of the subject being treated. Preferably, thecompounds of the present invention will be bind parasitic tubulin versusmammalian tubulin more selectively than oryzalin. Preferably, thecompounds of the present invention will also be less toxic to normalcells than oryzalin.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of improvement in disease severity and the frequency of incidence,while avoiding adverse side effects typically associated withalternative therapies.

The dosage form and amount can be readily established by reference toknown treatment regiments. The amount of therapeutically active compoundthat is administered and the dosage regimen for treating a diseasecondition caused by parasitic protozoa with the compounds and/orcompositions of this invention depends on a variety of factors,including the age, weight, sex, and medical condition of the subject,the severity of the disease, the route and frequency of administration,and the particular compound employed, the particular protozoa causingthe disease, as well as the pharmacokinetic properties of the individualtreated, and thus may vary widely. Such treatments may be administeredas often as necessary and for the period of time judged necessary by thetreating physician. One of skill in the art will appreciate that thedosage regime or therapeutically effective amount of the inhibitor to beadministrated may need to be optimized for each individual. Thepharmaceutical compositions may contain active ingredient in the rangeof about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mgand most preferably between about 1 and 200 mg. A daily dose of about0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

The dinitroaniline sulfonamide compounds of the present invention may beadministered by any suitable route known to those skilled in the art,preferably in the form of a pharmaceutical composition adapted to such aroute, and in a dose effective for the treatment intended. Thepharmaceutical composition comprises a therapeutically effective amountof a compound of Formula I or II, or a derivative or pharmaceuticallyacceptable salt or ester thereof in association with at least onepharmaceutically acceptable carrier, adjuvant, or diluent (collectivelyreferred to herein as “carrier materials”) and, if desired, other activeingredients. The active compounds of the present invention may beadministered by any suitable route known to those skilled in the art,preferably in the form of a pharmaceutical composition adapted to such aroute, and in a dose effective for the treatment intended( The activecompounds and composition may, for example, be administered orally,intra-vascularly, intraperitoneally, intranasal, intrabronchial,subcutaneously, intramuscularly or topically (including aerosol).

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are capsules, tablets, powders, granules or asuspension, with conventional additives such as lactose, mannitol, cornstarch or potato starch; with binders such as crystalline cellulose,cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators such as corn starch, potato starch or sodiumcarboxymethyl-cellulose; and with lubricants such as talc or magnesiumstearate. The active ingredient may also be administered by injection asa composition wherein, for example, saline, dextrose or water may beused as a suitable carrier.

For intravenous, intramuscular, subcutaneous, or intraperitonealadministration, the compound may be combined with a sterile aqueoussolution which is preferably isotonic with the blood of the recipient.Such formulations may be prepared by dissolving solid active ingredientin water containing physiologically compatible substances such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering said solution sterile. The formulations may be present in unitor multi-dose containers such as sealed ampoules or vials.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active compound which ispreferably made isotonic. Preparations for injections may also beformulated by suspending or emulsifying the compounds in non-aqueoussolvent, such as vegetable oil, synthetic aliphatic acid glycerides,esters of higher aliphatic acids or propylene glycol.

Formulations for topical use include known gels, creams, oils, and thelike. For aerosol delivery, the compounds may be formulated with knownaerosol excipients, such as saline, and administered using commerciallyavailable nebulizers. Formulation in a fatty acid source may be used toenhance biocompatibility.

The term “subject” for purposes of treatment includes any human oranimal subject who is affected with a disease caused by parasiticprotozoa, specifically Leishmania donovani, and also includingTrypanosoma cruzi, Trypanosoma brucie gambiense, Trypanosoma brucierhodesiense, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovali,and Plasmodium malariae. Such diseases include leishmaniasis, includingvisceral leishmaniasis, mucocutaneous leishmaniasis, and cutaneousleishmaniasis; Chagas disease; human African trypanosomiasis, also knownas African sleeping sickness; animal trypanosomiasis; and malaria.Besides being useful for human treatment, the compounds of the presentinvention are also useful for veterinary treatment of mammals, includingcompanion animals and farm animals, such as, but not limited to dogs,cats, horses, cows, sheep, and pigs. Preferably, subject means a human.

Derivatives are intended to encompass any compounds which arestructurally related to the dinitroaniline compounds of formulae I andII and have the ability to bind to parasite tubulin at least as well asoryzalin. In accordance with the present invention, the derivativesshould also bind to the parasitic tubulin with more affinity than tovertebrate tubulin. The derivatives should bind to the tubulin of one ormore of the parasites selected from Leishmania donovani, Trypanosomacruzi, Trypanosoma brucie gambiense, Trypanosoma brucie rhodesiense,Plasmodium falciparum, Plasmodium vivax, Plasmodium ovali, andPlasmodium malariae. By way of example, such compounds may include, butare not limited to, prodrugs thereof. Such compounds can be formed invivo, such as by metabolic mechanisms.

The present invention also relates to therapeutic methods of treatingpatients who have diseases caused by the above protozoa, which includeleishmaniasis, including visceral leishmaniasis, mucocutaneousleishmaniasis, and cutaneous leishmaniasis; Chagas disease; humanAfrican trypanosomiasis, also known as African sleeping sickness; animaltrypanosomiasis; and malaria The methods comprise administering atherapeutically effective amount of a compound of formula I, II or IIIto a subject having one of these disorders.

Also included in the family of compounds of formulae I, II and III arethe pharmaceutically acceptable salts thereof. The phrase“pharmaceutically acceptable salts” connotes salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically acceptable. Suitable pharmaceutically acceptable acidaddition salts of compounds of formulae I and II may be prepared from aninorganic acid or from an organic acid Examples of such inorganic acidsare hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric,and phosphoric acid. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids, examples of whichinclude formic, acetic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic,methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, galactaric,and galacturonic acids. Suitable pharmaceutically acceptable baseaddition salts of compounds of formulae I, II, and III include metallicsalts made from aluminum, calcium, lithium, magnesium, potassium,sodium, and zinc. Alternatively, organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine may be usedform base addition salts of the compounds of formulae I and II. All ofthese salts may be prepared by conventional means from the correspondingcompounds of formulae I, II, and III by reacting, for example, theappropriate acid or base with the compound of formula I, II, or III.

Where the term “alkyl” is used, it includes C₁ to C₁₀ linear or branchedalkyl radicals, examples include methyl, ethyl, propyl, isopropyl,butyl, tert-butyl, and so forth. The term “aryl” includes such aromaticradicals as phenyl, biphenyl, and benzyl, as well as fused aryl radicalssuch as naphthyl, anthryl, phenanthrenyl, fluorenyl, and indenyl and soforth. The term “aryl” also encompasses “heteroaryls,” which are arylsthat have carbon and one or more heteroatoms, such as O, N, or S in thearomatic ring. Examples of heteroaryls include indolyl, pyrrolyl, and soon. The term “alkylaryl” encompasses aryl radicals that have an alkylsubstituent at any substitutable position. The terms “haloaryl” and“haloalkyl” encompass aryls and alkyls, respectively, having one or morehalogen (F, Cl, Br, or I) atoms substituted to the aryl or allyl at anysubstitutable position.

All documents referenced herein are incorporated by reference.

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations.

References

-   1. World Health Organization. 2000,    www.who.int/inf-fs/en/fact116.html.-   2. Berman, J. D. Clin. Infect. Dis. 1997, 24, 684-703.-   3. Lira, R; Sundar, S.; Makharia, A.; Kenney, R; Gam, A.; Saraiva,    E.; Sacks, D. J. Infect. Dis. 1999, 180, 564-567.-   4. Morejohn, L.; Fosket, D. Pharmac. Ther. 1991, 51, 217-230.-   5. Chan, M. M.-Y.; Fong, D. Science 1990, 249, 924-926.-   6. Chan, M. M.-Y.; Grogl., M.; Chen, C.-C.; Bienen, E. J.; Fong, D.    Proc. Natl. Acad. Sci. USA 1993, 90, 5657-5661.-   7. Callahan, H. L.; Kelley, C.; Pereira, T.; Grogl, M. Antimicrob.    Agents Chemother. 1996, 40, 947-952.-   8. Werbovetz, K.; Brendle, J.; Sackett, D. Mol. Biochem. Parasitol.    1999, 98, 53-65.-   9. Benbow, J.; Bernberg, E.; Korda, A.; Mead, J. Antimicrob. Agents    Chemother. 1998, 42,339-343.-   10. Armson, A.; Kamau, S.; Grimm, F.; Reynoldson, J.; Best, W.;    MacDonald, L.; Thompson, R. Acta Tropica 1999, 73, 303-311.-   11. Chan, M. M.-Y.; Triemer, R. E.; Fong, D. Differentiation 1991,    46, 15-21.-   12. Schultz, H. In Organic Syntheses; Rabjohn, N., Ed.; John Wiley &    Sons, Inc.: New York, 1963; Collective Vol. IV, pp 364-366.-   13. Judkins, B.; Allen, D.; Cook, T.; Evans, B.; Sardharwala, T.    Synth. Comm. 1996, 26, 4351-4367.-   14. Havens, C.; Bryant, N.; Asher, L.; Lamoreaux, L.; Perfetto, S.;    Brendle, J.; Werbovetz, K. Mol. Biochem. Parasitol. 2000, 110,    223-236.-   15. Chan, M. M.-Y.; Tzeng, Y.; Emge, T. J.; Ho, C.-T.; Fong, D.    Antimicrob. Agents Chemother. 1993, 37, 1909-1913.-   16. Bai, R.; Lin, C.; Nguyen, N.; Liu, T.; Hamel, E. Biochemistry    1989, 28, 5606-5612.-   17. Shan, B.; Medina, J.; Santha, E.; Frankmoelle, W.; Chou, T.;    Learned, R; Narbut, M.; Stott, D.; Wu, P.; Jaen, J.; Rosen, T.;    Timmermans, P.; Beckmann, H. Proc. Natl. Acad. Sci. USA 1999, 96,    5686-5691.-   18. Morejohn, L.; Bureau, T.; Mole-Bajer, J.; Bajer, A.; Fosket, D.    planta 1987, 172, 252-264.

1. A compound of Formula I useful for the treatment of diseases causedby parasitic protozoa comprising:

wherein R¹ and R² can be the same or different and are selected from thegroup consisting of H and C₁-C₁₀ alkyl, provided that R₁ and R₂ are notboth n-propyl, or R₁ and R₂ can form a 5 or 6 membered ring containingC, N, and O; and R³ is a sulfonamide; wherein the nitrogen atom of R³can substituted with C₁-C₁₀ alkyl, aryl, alkylaryl, alkoxyaryl, orhaloaryl.
 2. The compound of claim 1 wherein R¹ and R² are selected thegroup consisting of methyl, ethyl, and butyl and combinations thereof.3. The compound of claim 1 wherein R¹ and R² are selected from the groupconsisting of morpholino or pyrrolidino.
 4. The compound of claim 1wherein the compound is useful for the treatment of diseases caused byparasitic protozoa, wherein the disease is visceral leishmaniasis,mucocutaneous leishmaniasis, cutaneous leishmaniasis, Chagas disease;human African trypanosomiasis, animal trypanosomiasis; or malaria. 5.The compound of claim 1 wherein the compound is less cytotoxic to normalcells than oryzalin.
 6. A compound of Formula II useful for thetreatment of diseases caused by parasitic protozoa comprising:

wherein R₄ and R₅ are the same and selected from the group consisting ofC₁-C₁₀ alkyl, or R₄ and R₅ form a pyrrolidine ring; and R₆ is selectedfrom the group consisting of C₁-C₁₀ alkyl, aryl, alkylaryl, alkoxyaryl,and haloaryl wherein the compound is less cytotoxic to normal cells thanoryzalin.
 7. The compound of claim 6 wherein R⁶ is selected from thegroup consisting of C₁-C₁₀ alkyl aryl, alkylaryl, alkoxyaryl, andhaloaryl.
 8. The compound of claim 7 wherein R⁶ is selected from thegroup consisting of methyl, ethyl, propyl, butyl, isopropyl, phenyl,methoxyphenyl, ethoxyphenyl, propoxyphenyl, methylphenyl, ethylphenyl,propylphenyl, butylphenyl, chloropheneyl, dichlorophenyl, fluorophneyl,difluorophenyl, and pyridnyl.
 9. The compound of claim 6 wherein R⁴ andR⁵ are selected from the group consisting of methyl, ethyl, propyl,butyl, pentyl, and hexyl.
 10. The compound of claim 6 wherein thecompound is useful for the treatment of diseases caused by parasiticprotozoa, wherein the disease is leishmaniasis, Chagas disease; humanAfrican trypanosomiasis, animal trypanosomiasis; or malaria.
 11. Acompound of Formula III useful for the treatment of diseases caused byparasitic protozoa comprising:

wherein n is 1, 2, 3, or 4; and Y is selected from the group consistingof NH₂, C₆H₅NH, C₆H₄(OCH₃)NH, C₆H₄(CH₃)NH, C₆H₄ClNH, provided that whenn=2 Y is not NH₂.
 12. The compound of claim 11 wherein the compound isselected from the group consisting of4-(dibutylamino)-3,5-dinitrobenzenesulfonamide;N1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methyl)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-Phenyl-3,5-dinitro-N4,N4-di-n-ethylsulfonilamide; andN1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide.
 13. The compound ofclaim 11 wherein the compound is used to treat diseases caused by L.donovani.
 14. The compound of claim 13 wherein the compound is selectedfrom the group consisting ofN1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide; andN1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide.
 15. The compound ofclaim 11 wherein the compound is used to treat diseased caused by T. b.brucei.
 16. The compound of claim 15 wherein the compound is selectedfrom the group consisting ofN1-Phenyl-3,5-dinitro-N4,N4di-n-propylsulfonilamide;N1-(3-Methoxy)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-(3-Methyl)phenyl-3,5-dinitro-N4,N4-di-n-propylsulfonilamide;N1-Phenyl-3,5-dinitro-N4,N4-di-n-ethylsulfonilamide; andN1-Phenyl-3,5-dinitro-N4,N4-di-n-butylsulfonilamide.
 17. The compound ofclaim 11 wherein the compound is less cytotoxic to normal cells thanoryzalin.
 18. A method of treating subjects having diseases caused byparasitic protozoa, the method comprising administering atherapeutically effective amount of a compound of claim 1, 6, or 11 to asubject in need of such treatment.
 19. The method of claim 18 whereinthe disease caused by parasitic protozoa is selected from visceralleishmaniasis, mucocutaneous leishmaniasis, and cutaneous leishmaniasis,Chagas disease; human African trypanosomiasis, animal trypanosomiasis;and malaria.
 20. The method of claim 19 wherein the disease caused byparasitic protozoa is selected from visceral leishmaniasis,mucocutaneous leishmaniasis, and cutaneous leishmaniasis.
 21. The methodof claim 16 wherein the subject is a person.